Cells are surrounded by plasma membranes. Plasma membranes contain components called glycosphingolipids inserted therein which aid in the formation of the characteristic surface structure of cells. Each type of cell is characterized by a specific profile of glycosphingolipid components, including those components known as gangliosides, located in its plasma membrane. Gangliosides contain a particular type of acidic carbohydrate known as sialic acid. Further, many specific types of cells, including tumor cells, are characterized by the presence of a particular type of ganglioside located in their plasma membranes.
In recent years, a number of monoclonal antibodies have been established after immunization with human tumor cells or tissues. The monoclonal antibodies were selected by positive reactivity with tumor cells and negative reactivity with normal cells or tissues. Many monoclonal antibodies selected for preferential reactivity with melanomas, neuroblastomas and adenocarcinomas have been identified are directed to gangliosides.
Some of the anti-ganglioside antibodies with specific isotypes (particularly IgG.sub.3 and IgG.sub.2a) and which show strong reactivity with gangliosides suppress tumor growth in vivo. For example, melanomas of some patients regressed following large dose administration of a specific anti-GD.sub.3 ganglioside antibody (Houghton, A. N. et al., Proc. Natl. Acad. SCi. USA, 82:1242-1246 (1985)).
Further, it was demonstrated recently that GM.sub.2 adsorbed on BCG bacteria showed a detectable immune response. Thus, it was asserted that GM.sub.2 could be a useful vaccine for human melanomas (Livingston, P.O. et al., Proc. Natl. Acad. Sci. USA, 84:2911-2915 (1987)).
Hence, gangliosides are important antigens and immunogens of tumor tissues and cells (Hakomori, S., Ann. Rev, Immunol., 2:103-126 (1984); Hakomori, S., In Handbook of Lipid Research, Volume 3, Sphingolipid Biochemistry, Kanfer, J. N. et al., Eds., Plenum, New York, pages 1-165 (1983); and Hakomori, S., Sci. Amer., 254:44-53 (1986)).
However, although gangliosides are important cell type-specific markers, gangliosides are poor immunogens for eliciting humoral or cellular immune responses.
A small portion of gangliosides are present in tumor cells and tissues in the form of a lactone thereof. For example, less than 0.1% of the particular ganglioside, designated GM.sub.3, present in melanoma cells was identified as a lactone thereof.
Ganglioside lactones are defined as the inner ester between the carboxyl group of the sialic acid and the primary or secondary hydroxyl group of the sugar residues within the same molecule.
While galactoside lactones have been detected and are believed to occur naturally as plasma membrane components, the quantity thereof is extremely low and thus the natural occurrence thereof is disputed (Nores, G. A. et al., J. Immunol., 139:3171-3176 (1987) and Riboni, L., J. Biol. Chem., 261:8514-8519 (1986)).
Despite the question about the natural occurrence of said lactones, it was demonstrated that ganglioside lactones are strong immunogens which can cause a much greater immune response than native gangliosides (Nores, G. A. et al., J. Immunol., 139:3171-3176 (1987)). Further, Nores et al., supra, found that the antibodies produced using ganglioside lactones as immunogens often are of the IgG.sub.3 isotype.
In the present invention, it was found that the use of tumor cells and tumor cell membranes followed by a booster with purified ganglioside lactones in the immunization step of producing hybridomas provides new hybridomas that produce monoclonal antibodies (mAb's) directed to a variety of tumor-associated gangliosides, including human cancer-associated fucogangliosides. Further, while some of the new antibodies show similar binding specificity to known antibodies, the isotypes thereof are different. Therefore the pharmacodynamic activities thereof also are expected to be different. Further, many of the new antibodies have unique cross-reactivity.
Monoclonal antibodies directed to lacto-series type 2 chain structures such as i, Le.sup.x Le.sup.y and , sialosyl-Le.sup.x are known to react with a large number of common human cancers derived from tissues of lung, breast, stomach, colon, liver, pancreas and other endodermal epithelial sources. For example, Hoff et al., Canc. RES., 49:6883-6888 (1989) and Inafusa et al., Clin. Exp. Metas., 9:245-257 (1991) found high expression of sialyl Le.sup.x in metastatic tumors.
Some of the tumor-associated carbohydrate antigens (TACA's) also are expressed in normal mature myelocytes and monocytes, as well as in leukemic leukocytes. However, it is unknown whether those TACA's are expressed in hematopoietic cells or progenitors and in leukemic leukocytes. Data on expression of those TACA's in hematopoietic progenitor cells is desirable since mAb's directed to type 2 chain-based structures could serve as useful clinical reagents for targeting leukemic leukocytes as well as solid tumors if the antigens are not expressed or are expressed at lower levels in normal or progenitor cells.